Analysis of the honeybee sperm proteome using MudPit and 2D PAGE

Honeybee semen was collected by gently squeezing the male abdomens. Samples were pooled and then centrifuged. Pelleted sperm was collected and analysed with both 2D PAGE and gel-free methods. For the 2D PAGE, protein spots were digested using trypsin. Extracted peptides, resolved on a C18 column, were analysed by an Agilent LC/MSD Trap XCT Ultra 6330 mass spectrometer. Spectra were searched against the honeybee protein sequences (RefSeq release 48) using Mascot algorithm (with 1 missed cleavage, Cys-carbamidomethylation as fixed modification, and Met-oxidation and N/Q-deamidation as variable modifications). For the MudPit analysis the sperm sample was digested by trypsin. Peptides were resolved using strong cation exchange chromatography followed by reverse phase (C18) HPLC and finally analysed by an Agilent QTOF mass spectrometer. Resulting spectrum files were converted to mzXML and merged using ProteoWizard msconvert. These were then searched against honeybee protein sequences using Mascot, Omssa and X!tandem (all with 1 missed cleavage, and Met-oxidation and N/Q-deamidation as variable modifications). Results of the three search engines were pooled using TPP.